333 - Regulation of human sirtuin deacylase activity by cellular oxidants

Abstract

Sirtuins catalyze the NAD+-dependent deacylation of acyl-lysine residues, producing O-acyl-ADP-ribose and nicotinamide. Humans encode seven sirtuins (Sirt1-7) that are considered pro-survival proteins, and decreased sirtuin activity promotes aging-related diseases, including type-II diabetes. However, how sirtuin activity is inhibited during aging is largely unknown. We are defining the physiological mechanisms that regulate sirtuin activity by post-translational modifications, with the long-term goal to exploit these mechanisms to prevent diseases associated with decreased sirtuin activity. As oxidative stress increases with age, our current focus is on post-translational regulation of sirtuin activity by cellular oxidants. Here, we show that Sirt1 can be nitrosated by S-nitrosoglutathione (GSNO). Colorimetric Zn2+ loss and circular dichroism assays revealed Sirt1 nitrosation correlated with Zn2+-release and loss of α-helical structure, suggesting the target of nitrosation is the Zn2+-tetrathiolate conserved among sirtuins. Molecular dynamics simulations suggested Zn2+ loss due to Sirt1 nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, disrupting NAD+and acetyl-lysine substrate binding. Furthermore, Sirt1 nitrosation was reversed upon exposure to thiol-based reducing-agents, resulting in restoration of Sirt1 activity. This restoration was dependent on the presence of Zn2+, consistent with nitrosation of the Zn2+-tetrathiolate as the source of Sirt1 inhibition. More recently, we found that nuclear sirtuins (Sirt1, Sirt2, Sirt6) are more broadly inhibited by nitric oxide (NO) and NO-derived oxidants, and are resistant to inhibition by oxidized glutathione, hydrogen peroxide, and hydrogen sulfide. Surprisingly, mitochondrial sirtuins (Sirt3 and Sirt5) displayed selective inhibition by peroxynitrite and were insensitive to all other assayed oxidants. These data suggest that, despite conservation of the Zn2+-tetrathiolate across all sirtuins, nitrosation does not universally inhibit sirtuins. Additionally, we observed a concentration- and time-dependent increase in Sirt6 expression in pancreatic beta cells in response to NO, suggesting that NO modulates Sirt6 expression in beta cells under inflammatory conditions.